Detection and treatment of intravascular lesions

ABSTRACT

Optical agents that contain a fibrin binding moiety covalently linked to an optical dye are described, as well as methods of treating intravascular lesions in a patient using such optical agents.

TECHNICAL FIELD

This invention relates to compositions and methods for the detection andtreatment of intravascular lesions, and more particularly to the use ofoptical agents in conjunction with medical devices to treatintravascular lesions.

BACKGROUND

Cardiovascular disease is a primary health threat in the developedworld. Certain intravascular lesions, such as deep vein thrombosis,pulmonary embolism, and atherosclerotic plaques, are clinicalmanifestations of cardiovascular disease that have significant morbidityand mortality profiles. For example, in the United States alone, thereare an estimated 600,000 patients that suffer pulmonary embolism eachyear. Approximately 114,000 of these patients later die due tocomplications associated with the disease.

The high mortality rate is partly due to significant limitationsassociated with currently available methods to detect intravascularlesions. In particular, identification of intravascular lesions iscomplicated because of their very location in blood vessels. Blood is aflowing, non-transparent mixture of protein and cells, the net effect ofwhich is a significant background that interferes with detection. As aresult, many methods to detect intravascular lesions are inconclusive.In addition, many methods for detection require a time frame thatfunctionally prevents the administration of a treatment in a clinicallyeffective time period.

It would be useful to have a method to treat intravascular lesions thatcombines sensitive detection of the lesions with immediate access to atherapy designed to reduce the size or to alter the shape of the lesion.

SUMMARY

This invention relates to compositions and methods for the detection andtreatment of intravascular lesions, and more particularly to the use ofoptical agents in conjunction with medical devices to treatintravascular lesions. The use of the methods and compositions of thepresent invention enhances the sensitivity and facilitatesadministration of therapies in a timely fashion. In addition, themethods allow real-time monitoring of the therapy to determine aclinically effective endpoint at which to stop the therapy.

Accordingly, one aspect of the invention provides a method for treatingan intravascular lesion in a patient. The term “intravascular lesion”means a lesion within a blood vessel. For example, the lesion can be athrombus, a clot, an atherosclerotic plaque, or an embolus. The lesionmay include fibrin that is exposed to blood flowing in the blood vessel.The method includes administering an optical agent (e.g., orally orparenterally such as intravenously, intraarterially, interstitially,intrathecally, subcutaneously, or intracavity), wherein the opticalagent includes a fibrin binding moiety and an optical dye, and whereinthe optical agent can form a fibrin-optical agent complex at the site ofthe lesion. A signal from the fibrin-optical agent complex is detectedusing a device inserted near the lesion and data is obtained about thelesion based on the signal of the fibrin-optical agent complex. Atherapy is then delivered, based on the obtained data, to at least aportion of the lesion, e.g., so that the size of the lesion is reducedor the shape of the lesion is altered.

The fibrin binding moiety may include a peptide. For example, the fibrinbinding moiety may include the amino acid sequenceCys-Asp-Tyr-Tyr-Gly-Thr-Cys (SEQ ID NO: 1), the amino acid sequenceCys-Pro-Tyr-Xaa-Leu-Cys (SEQ ID NO:2), where Xaa can be Gly or Asp, orthe amino acid sequence Cys-Hyp-Tyr(3×)-Xaa-Leu-Cys (SEQ ID NO:3), where3× represents a halogen, nitro-, or trifluoromethyl group at the 3position of the benzyl ring of the Tyrosine, where Hyp representsHydroxyproline, and where Xaa can Gly or Asp. The fibrin binding moietyalso can include the amino acid sequencePhe-His-Cys-Hyp-Tyr(3-I)-Asp-Leu-Cys-His-Ile-Leu (SEQ ID NO:4), whereTyr(3-I) represents 3-iodo-tyrosine and Hyp represents Hydroxyproline.

In some embodiments, the optical dye is covalently bound to theN-terminal amino acid of a peptide fibrin-binding moiety. The N-terminalamino acid can be a naturally-occurring or a non-naturally-occurringamino acid. For example, the N-terminal amino acid can be β-alanine(β-ala), 6-aminohexanoic acid (Ahx), or a lysine residue. The C-terminusof the fibrin binding moiety's amino acid sequence may be capped as aC-terminal amide. Alternatively, the C-terminus may be capped with anon-optical moiety. The C-terminal amino acid also can be in theD-configuration.

In other embodiments, the optical dye is covalently bound to theC-terminal amino acid of a peptide fibrin binding moiety. The N-terminusof the fibrin binding moiety's amino acid sequence may be alkylated. TheN-terminal amino acid also can be in the D-configuration.

The optical dye can be selected from the group consisting offluorescein, rhodamine, tetramethylrhodamine, hematoporphyrin,fluoresdamine, indocyanine, tetramethylrhodamine, Cosin, erythrosine,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue, Texas Red, and derivatives thereof. In oneembodiment, the optical dye is fluorescein. In another embodiment, theoptical dye is tetramethylrhodamine.

Specific embodiments of optical agents for use in the method of thepresent invention include:

In one embodiment, the dissociation constant of the optical agent has avalue less than about 10 μM. In another embodiment, the dissociationconstant value of the optical agent is less than about 5 μM.Alternatively, the dissociation constant value of the optical agent isless than about 1 μM. The dissociation constant of the optical may alsobe less than about 0.3 μM.

The device inserted near the lesion may include a catheter and anoptical detector, such as a fluorescence emission detector. The devicemay further include an excitation source. The device can be insertednear the lesion, in a cavity, a tissue, an interstitial space, or ablood vessel. In one embodiment, the device is inserted in the sameblood vessel as the lesion.

The therapy can include a thrombolytic composition, such as tissueplasminogen activator (tPA), streptokinase, antistreplase, or urokinase.Alternatively, the therapy can include a mechanical manipulation of thelesion, such as by balloon angioplasty. In another embodiment, thetherapy can include laser ablation of the lesion.

The therapy can be delivered by the device inserted near the lesion.Alternatively, in an embodiment where the therapy is a thrombolytic, thetherapy can be administered intravenously at a site remote from thelesion. The therapy is delivered to at least a portion of the lesion. Inone embodiment, the thrombolytic agent is delivered to about 90% of thesurface of the lesion. In another embodiment, the thrombolytic isdelivered to about 50% of the surface of the lesion. In yet anotherembodiment, the thrombolytic is delivered to about 10% of the surface ofthe lesion.

The method can include detecting the signal of the fibrin-optical agentcomplex during the delivery of the therapy. The method can includestopping the therapy delivery when the signal of the fibrin-opticalagent complex decreases to a predetermined value. For example, in oneembodiment, the therapy is stopped when the signal of the fibrin-opticalagent complex is less than about 90% of the signal before delivery ofthe therapy. In another embodiment, the therapy is stopped when thesignal of the fibrin-optical agent complex is less than about 50% of thesignal before delivery of the therapy. In yet another embodiment, thetherapy is stopped when the signal of the fibrin-optical agent complexis less than about 10% of the signal before delivery of the therapy.

In another aspect, the invention features compositions and kits thatinclude an optical agent, wherein the optical agent includes an opticaldye covalently linked to the N-terminus of a peptide fibrin bindingmoiety (FBM) via a linker, wherein the optical agent has the generalformula:

Particular embodiments of optical agents include structures I-XIII andpharmaceutically acceptable salts thereof.

The invention also features formulations that include compositionscontaining optical agents, wherein the formulation includes at least oneingredient selected from the group consisting of solubilizing agents,excipients, carriers, adjuvants, vehicles, preservatives, a localanesthetic, flavorings, and colorings.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, themethods, materials, and examples are illustrative only and not intendedto be limiting.

Commonly used chemical abbreviations that are not explicitly defined inthis disclosure may be found in The American Chemical Society StyleGuide, Second Edition; American Chemical Society, Washington, D.C.(1997); “2001 Guidelines for Authors,” J. Org. Chem. 66(1), 24A (2001);and “A Short Guide to Abbreviations and Their Use in Peptide Science,”J. Peptide Sci. 5, 465-471 (1999).

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a table demonstrating the structures of embodiments of opticalagents with their dissociation constants (Kd) to a DD(E) fragment offibrin at 24° C.

FIG. 2 demonstrates a general synthetic scheme to couple an optical dyeto the fibrin binding moieties of the present invention.

FIG. 3 provides the structures of two optical agents used in the imagingstudies of Example 2.

DETAILED DESCRIPTION

The invention provides optical agents and methods for detecting andtreating intravascular lesions using the optical agents. Optical agentsof the invention include an optical dye (OD) linked to a fibrin bindingmoiety (FBM), and have affinity for fibrin. After administration of anoptical agent to a mammal (e.g., a human patient), the optical agent canform a fibrin-optical agent complex, which has a detectable signal,allowing for improved sensitivity of lesion detection. The affinity forfibrin is useful because fibrin is present in most lesions and can betargeted without interfering with normal thrombolytic processes.

The improved sensitivity allows for the detection of relatively smalllesions and provides information about the presence and distribution offibrin in the lesion. The use of optical agents and medical devices(e.g., a catheter) inserted near the lesion to detect the signal offibrin-optical agent complexes also avoids the interference due to thebackground of flowing blood in a blood vessel. The medical devicesinserted near the lesion may be used to deliver a therapy to at least aportion of the lesion in a timely and effective manner in order toreduce the size of the lesion or to alter the shape of the lesion. Bymonitoring the signal of the fibrin-optical agent complexes during thecourse of the therapy, the therapy may be stopped at a clinicallysignificant timepoint.

Optical Agents

Optical agents of the invention include an OD and a FBM covalently boundto each other, either directly or via a linker (OD-L-FBM). The FBM canbe a small molecule or a peptide. As used herein, the term “peptide”refers to a chain of amino acids that is about 2 to about 75 amino acidsin length (e.g., 3 to 50 amino acids). Affinity of a peptide for fibrincan be expressed in terms of its dissociation constant (Kd), which isthe equilibrium constant for the dissociation reaction of the peptidefrom the DD(E) fragment of fibrin. The term “DD(E) fragment of fibrin”refers to a fibrin subcomponent generated by proteolytic degradation offibrin with plasmin or trypsin. The DD(E) fragment is a complex of thecrosslinked D domains of adjacent fibrin monomers with the central Edomain of fibrin (See, for example, Spraggon et al., Nature 389:455462(1997)). Since DD(E) is a product resulting from the proteolysis offibrin, one of skill in the art will understand that there may be someslight heterogeneity in its composition. The DD(E) fragment can bebiotinylated and immobilized via avidin to a solid substrate (e.g., amulti-well plate). Peptides can be incubated with the immobilized DD(E)fragment in a suitable buffer and binding detected using knownmethodology. Methods for determining the dissociation constant of thepeptide for DD(E) are set forth in WO 01/09188.

As a result of the FBM having affinity for fibrin, the optical agentalso has affinity for fibrin. The term “affinity” refers to the capacityof the optical agent to be taken up by, retained by, or bound to thefibrin in the lesion. The affinity of an optical agent can be expressedin terms of its Kd, which is the equilibrium constant for thedissociation reaction of the optical agent from fibrin, and determinedas discussed above for the peptide. The dissociation constant of theoptical agent for DD(E) can have a value less than about 10 μM (e.g.,0.1 μM to 10 μM). In one embodiment, the dissociation constant value isless than about 5 μM. In another embodiment, the dissociation constantvalue is less than about 1 μM. The dissociation constant also may have avalue less than about 0.3 μM (e.g., 0.2 μM).

Peptide fibrin binding moieties can include naturally occurring ornon-naturally occurring amino acids. As used herein, the term “natural”or “naturally occurring” amino acid refers to one of the twenty mostcommon occurring amino acids. Natural amino acids are referred to bytheir standard one- or three-letter abbreviations. The term “non-naturalamino acid” or “non-natural” refers to any derivative of a natural aminoacid including D forms, β and γ amino acid derivatives, α-N-alkylatedamino acids, and amino acids having amine-containing side chains (suchas Lys or Orn) in which the amine has been acylated or alkylated. It isnoted that certain amino acids, e.g., hydroxyproline, that areclassified as a non-natural amino acid herein, may be found in naturewithin a certain organism or a particular protein.

The fibrin binding moieties of the present invention may be cyclized oruncyclized. When cyclized, the fibrin binding moieties have a disulfidelinkage between two cysteine residues in their amino acid sequence.Cyclization can occur using known methods, either before, during, orafter modification of the FBM with the optical dye. See FIG. 2.

In some embodiments, the C or N-terminus of a fibrin binding moiety'samino acid sequence can be capped. For example, the C-terminus can becapped with an amide or the N-terminus can be capped by alkylating theamine group. Alternatively, the C or N-terminus can be capped using anynon-optical moiety. The term “non-optical moiety” as used herein refersto any molecule that is not an optical dye. When the C or N-terminus isso capped, the optical agents of the present invention include oneoptical dye molecule per optical agent molecule. While not being boundby any theory, when the optical agent has only one optical dye moleculeper optical agent molecule, the possibility of intramolecular quenchingof the optical signal from one optical dye molecule to another opticaldye molecule on the same optical agent molecule is eliminated.

The C and N-termini also can be rendered less susceptible todegradation, e.g., degradation by metabolic and proteolytic processes.For example, the C or N-terminal amino acid of the FBM may be a D-aminoacid (i.e., having the “D” stereochemistry) in order to stabilize theFBM against degradation by proteases.

A FBM of the invention can include the amino acid sequenceCys-Asp-Tyr-Tyr-Gly-Thr-Cys (SEQ ID NO:1) or Cys-Pro-Tyr-Xaa-Leu-Cys(SEQ ID NO:2), wherein Xaa can be Gly or Asp. In another embodiment, thefibrin binding moiety includes the amino acid sequenceCys-Hyp-Tyr(3×)-Xaa-Leu-Cys (SEQ ID NO:3), where 3× represents ahalogen, nitro-, or trifluoromethyl group at the 3 position of thebenzyl ring of the Tyrosine, Hyp represents Hydroxyproline (e.g.,4-hydroxyproline), and Xaa is Gly or Asp. In yet another embodiment, thefibrin binding moiety includes the amino acid sequencePhe-His-Cys-Hyp-Tyr(3-I)-Asp-Leu-Cys-His-Ile-Leu (SEQ ID NO:4), whereTyr(3-I) represents 3-iodo-tyrosine and Hyp represents Hydroxyproline.

Peptide fibrin binding moieties can be synthesized using known peptidesynthesis methods, including solid phase synthesis. Amino acids withmany different protecting groups appropriate for immediate use in solidphase synthesis of peptides are commercially available. Example 1demonstrates the synthesis of a FBM using a solid phase synthesismethod. Additional methods and details for the synthesis of the fibrinbinding moieties may be found in WO 01/09188.

Once synthesized, a FBM can be covalently coupled to an optical dye. Forexample, the optical dye can be covalently bound to the N-terminal aminoacid of a FBM (e.g., a β-alanine, 6-aminohexanoic acid, or lysineresidue, see FIG. 1), the C-terminal amino acid of a FBM (e.g., aleucine residue), or to both the N and C-termini of a FBM. In someembodiments, it is preferred that when the C-terminus is linked to anOD, the N-terminus is not covalently bound to or capped with an OD. TheOD provides an optical signal that allows the FBM to be detected (e.g.,by a fluorescence emission spectrum). Any OD may be used, provided itdoes not render the optical agent pharmaceutically unacceptable.Non-limiting examples of suitable OD include fluorescein, rhodamine,hematoporphyrin, fluoresdamine, indocyanine, tetramethylrhodamine,Cosin, erythrosine, coumarin, methyl-coumarins, pyrene, Malacite green,stilbene, Lucifer Yellow, Cascade Blue®, Texas Red® (Molecular Probes,Inc., Eugene, Oreg.), and derivatives thereof. Fluorescein andtetramethylrhodamine are particularly useful ODs. FIG. 2 demonstrates amethod for the modification of a FBM with the optical dye fluorescein.

In some embodiments, the OD is covalently linked to a FBM via a linker.Suitable linkers can be peptidic or non-peptidic in nature, and can bean all-carbon chain, or can contain heteroatoms such as, e.g., oxygen,nitrogen, sulfur, and phosphorus. The linker can be a linear or branchedchain, or can include structural elements such as phenyl ring(s),non-aromatic carbocyclic or heterocyclic ring(s), double or triplebond(s), and the like. Linkers may be substituted with alkyl, aryl,alkenyl, or alkynyl groups.

In some embodiments, a linker can have one of the following formulas:

Optical agents of the invention may contain one or more asymmetriccarbon atoms and thus may occur as racemates and racemic mixtures,single enantiomers, diastereomeric mixtures, and individualdiastereomers. All such isomeric forms of these compounds are includedin the present invention. Although the specific compounds exemplified inthis application may be depicted in a particular stereochemicalconfiguration, compounds having either the opposite stereochemistry atany given chiral center or mixtures thereof are also envisioned.

Specific embodiments of optical agents for use in the method of thepresent invention are shown in FIG. 1. In FIG. 1, “fluor” indicates afluorescein as the OD; Ahx indicates 6-aminohexanoic acid; β-alaindicates β-alanine; P(4-OH) indicates Hydroxyproline (Hyp); and Y(3-I)indicates 3-Iodo-Tyrosine. Single capital letters in the tablecorrespond to the single amino acid letter code. The NH2 in position 13indicates that the C-terminus of amino acid number 12 is capped as anamide. FIG. 1 provides the Kd (μM) vs. DD(E) for each optical agent.

Specific structures corresponding to FIG. 1 include:

Additional examples of optical agents include Structures XII and XIII(shown below). In structures XII and XIII, the OD is a coumarin dye. TheKd vs. DD(E) of structure XII is 6.6 μM; the Kd vs. DD(E) of structureXIII is 0.2 μM.

Methods of Treating Intravascular Lesions

According to one aspect of the invention, a method is provided to treatan intravascular lesion. The term “intravascular lesion” means a lesionwithin a blood vessel. “Blood vessel” as used herein can includearteries, veins, capillaries, and chambers of the heart. The lesion canbe a thrombus, a clot, an atherosclerotic plaque, or an embolus. Inparticular, the lesion can be a deep vein thrombus, a coronary thrombus,a carotid thrombus, an atherosclerotic plaque, including plaquecharacterized as high risk, an atrial or ventricular thrombus, an aorticarch thrombus, or a pulmonary embolus.

The lesion may include fibrin on its surface. The exposed fibrin may bein contact with blood flowing in the blood vessel. While not being boundby any theory, it is believed that the optical agent can form afibrin-optical agent complex more efficiently when there is exposedfibrin on the lesion's surface. In addition, while again not being boundby any theory, it is believed that lesions with exposed fibrin are atthe highest risk for spontaneous dislodging.

The method includes administering an optical agent or a derivativethereof. Suitable optical agent derivatives include any pharmaceuticallyacceptable salt, ester, or other derivative of a composition of thisinvention, which, upon administration, is capable of providing (directlyor indirectly) a compound of this invention or an active metabolite orresidue thereof. Other derivatives are those that increase thebioavailability of the compounds when administered or which enhancedelivery to a particular biological compartment.

Optical agents or derivatives thereof are formulated in apharmaceutically acceptable manner such that the agent can beadministered to a patient or animal without unacceptable adverse effectsThe optical agent can be formulated in accordance with routineprocedures as a pharmaceutical formulation adapted for human patients oranimals. Where necessary, the formulation can include such ingredientsas solubilizing agents, excipients, carriers, adjuvants, vehicles,preservatives, a local anesthetic, flavorings, colorings, and the like.The ingredients may be supplied separately, e.g., in a kit, or mixedtogether in a unit dosage form. The dosage to be administered and themode of administration will depend on a variety of factors includingage, weight, sex, condition of the patient, pharmacokinetic parametersof the formulation, genetic factors, and the like. As one of skill inthe art will recognize, the dosage will ultimately decided by theclinician.

The optical agent can be administered in any number of conventionalways, including orally or parenterally (e.g., subcutaneously,intravenously, intraarterially, interstitially, intrathecally, orintracavity administration). After administration, the optical agentforms a fibrin-optical agent complex at the site of the lesion. Theaffinity of the optical agent for fibrin allows the agent to localize atthe fibrin within or on the lesion. The fibrin-optical agent complexeshave an optical signal that can be detected. For example, the opticalsignal may be a fluorescence emission spectrum. The optical signal fromthe fibrin-optical agent complexes may be the same as or different fromthe optical signal of the optical agent before administration. Forexample, the signal may undergo a shift, a reduction, or an enhancementin a fluorescence wavelength maximum. The signal can be any opticalsignal that can be detected, including transmission or absorption of aparticular wavelength of light, fluorescence or phosphorescenceabsorption and emission, reflection, changes in absorption amplitude ormaxima, and elastically scattered radiation.

A device is inserted near the lesion to obtain information (i.e., data)about the lesion based on detecting a signal of the fibrin-optical agentcomplexes. A catheter such as the OPTICATH® family of fiber-opticcatheters sold by Abbott Laboratories can be used for detecting thesignal from the fibrin-optical agent complex. These catheters also canoptionally be used for delivering the therapy to the lesion. Otherpossible fiber-optic catheters can be obtained from COOK and BaxterHealthcare corporations. Fiber-optic catheters from the WellmanLaboratories of Photomedicine at the Massachusetts General Hospital aresuitable for detecting the fibrin-optical agent complex. FISOTechnologies has high quality fiber-optic sensors designed for insertioninto catheters. Other possible fiber-optic catheter detection systemsare disclosed in U.S. Pat. Nos. 4,175,545; 4,416,285; 4648,892;5,015,463; and 6,366,726.

The general position of the lesion typically is determined before thedevice is inserted nearby. The general position of the lesion may bedetermined by detecting the fibrin-optical agent complexes with adetector outside of the body of the patient. For example, if the opticalagent includes a fluorescent optical dye, the location of thefluorescence emission of the optical dye, which generally corresponds tothe location of the fibrin-optical agent complexes, may be determinedwith a fluorescence detector located outside the body of the patient.Alternatively, the position for device insertion is determined byreference to any number of known methods to determine the generallocation of a lesion, including the use of magnetic resonance orradionuclide-labeled agents that target a lesion, X-ray angiographictechniques, ventilation-perfusion scans of the lungs, and the like.

In another embodiment, the general position of the lesion may bedetermined by knowledge of the location where a lesion has occurred inthe past. For example, the general position of a lesion can be estimatedby reference to the medical history of the patient, e.g., the locationwhere a stent or angioplastic procedure had been performed in the past,or the location where the patient is known to have experienced lesionsin the past.

The device is inserted near the lesion. The device may be inserted intoa cavity, a tissue, an interstitial space, or a blood vessel. In oneembodiment, the device is inserted in the same blood vessel as thelesion. For example, if the device is a catheter, the catheter may beplaced within 10 cm of the lesion. Alternatively, the catheter may beplaced within 5 cm of the lesion. Alternatively, the catheter may beplaced within 1 cm of the lesion.

Information about the lesion is based on detecting a signal of thefibrin-optical agent complex. The device inserted near the lesion mayinclude an optical detector to detect the signal of the fibrin-opticalagent complex. In one embodiment, the device includes a fluorescenceemission detector. The device can also include an excitation source. Theexcitation source can provide the excitation wavelength of light, ifnecessary, to result in the optical signal generated by thefibrin-optical agent complex and detected by the optical detector. Forexample, excitation of the optical dye fluorescein occurs at 492 nm,while emission is detected at 515 nm. Excitation of the optical dyetetramethylrhodamine occurs at 555 nm, while emission is detected at 575nm.

The information about the lesion that is obtained based on the detectionof the signal of fibrin-optical agent complexes can include the size andshape of the lesion; the surface features of the lesion; thedistribution and relative amount of fibrin within the lesion, includingthe amount exposed on the surface of the lesion; an assessment of therisk profile (e.g., ability to dislodge spontaneously) of the lesion;and an estimate of vessel occlusion and stenosis.

After the information about the lesion is obtained, a therapy isdelivered based on the obtained information to at least a portion of thelesion. The therapy can be delivered by the device inserted near thelesion. For example, if the device is a catheter, the catheter candeliver the therapy to the lesion nearby. Alternatively, in anembodiment where the therapy is a thrombolytic, the therapy can bedelivered intravenously at a site remote from the lesion. In oneembodiment the therapy is delivered to about 90% of the surface of thelesion. In another embodiment, the therapy is delivered to about 50% ofthe surface of the lesion. In yet another embodiment, the therapy isdelivered to about 10% of the surface of the lesion.

The therapy should either reduce the size of the lesion or alter theshape of the lesion. To reduce the size of the lesion, the therapy caninclude a thrombolytic composition such as tissue plasminogen activator(tPA), streptokinase, antistreplase, or single or two-chain urokinase.Additional information concerning the use of thrombolytics, includingdosage, formulation, and time course of treatment are set forth in WO01/09811.

To reduce the size of the lesion or to alter its shape, the therapy caninclude a mechanical manipulation of the lesion. For example, theinserted device can include components to perform a balloon angioplastyat the site of the lesion. Balloon angioplastic treatment of a lesioncan reduce the size of the lesion or alter its shape by flattening itagainst the blood vessel to allow blood flow. Angioplasty, sometimescalled “PTCA” (percutaneous transluminal coronary angioplasty)represents the majority of interventional procedures. In this procedure,a catheter is inserted near the site of the lesion, and a tiny balloonis inflated. These devices compress the lesion against the artery wall,and open the artery, thus allowing increased flow. See, for example,Kandarpa K, et al., “Transcatheter interventions for the treatment ofperipheral atherosclerotic lesions: part II,” Journal of Vascular &Interventional Radiology. 12(7):807-12 (2001 July); Kandarpa K, et al.,“Transcatheter interventions for the treatment of peripheralatherosclerotic lesions: part I,” Journal of Vascular & InterventionalRadiology. 12(6):683-95 (2001 June).

In another embodiment, the therapy can include laser ablation of thelesion. For example, the inserted device can include components toperform laser ablation at the site of the lesion. See for example, theweb site of the American Heart Organization (Heart and Stroke A to ZGuide), wherein it is noted that: “laser angioplasty is a technique usedto open coronary arteries blocked by plaque (the build-up of cholesteroland other fatty substances in the inner lining of an artery). A catheter(thin tube) with a laser at the tip is inserted into an artery andadvanced through the blood vessels to the blocked artery in the heart.The laser emits pulsating beams of light that vaporize the plaque. Thisprocedure has been used alone and with balloon angioplasty.”

The method can further include detecting the signal of thefibrin-optical agent complex during the delivery of the therapy. As thetherapy is delivered, the inserted device continues to detect the signalof the fibrin-optical agent complex. The clinician can determine, basedon the signal detected, when to stop delivery of the therapy. The methodcan include stopping the therapy delivery when the signal of thefibrin-optical agent complex decreases to a predetermined value. Forexample, in one embodiment, the therapy is stopped when the signal ofthe fibrin-optical agent complex is less than about 90% of the signalbefore delivery of the therapy. In another embodiment, the therapy isstopped when the signal of the fibrin-optical agent complex is less thanabout 50% of the signal before delivery of the therapy. In yet anotherembodiment, the therapy is stopped when the signal of the fibrin-opticalagent complex is less than about 10% of the signal before delivery ofthe therapy.

In the methods of the present invention, the optical agent forms afibrin-optical agent complex at the site of the lesion. The ability ofthe optical agent to form a fibrin-optical agent complex may be measuredby examining the optical agent's dissociation constant (Kd) for a DD(E)fragment of fibrin as discussed above.

Articles of Manufacture

Optical agents described herein can be combined with packaging materialand sold as articles of manufacture or kits. Components and methods forproducing articles of manufactures are well known. The articles ofmanufacture may combine one or more optical agents described herein. Inaddition, the articles of manufacture may further include one or more ofthe following: sterile water or saline, pharmaceutical carriers,buffers, syringes, or catheters. A label or instructions describing howthe optical agent can be used for treating an intravascular lesion maybe included in such kits. The optical agents may be provided in apre-packaged form in quantities sufficient for single or multipleadministrations.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Synthesis of Optical Agents

Preparation of Fibrin Binding Moiety-Solid Phase Synthesis

NovaSyn TGR resin (0.20 mmol/g, 100 mg, 20 μmol) was washed withNMP/ether/NMP. The peptide was assembled by the standard solid phasemethod using the PyBOP/HOBt/DIEA activation. After the coupling of thefinal amino acid residue, the resin bound peptide was treated with asolution of piperidine in DMF (20% by volume, 2.0 mL) for 10 minutes toremove the Fmoc protecting group. The resin was washed thoroughly withNMP/ether/NMP, and was treated with a solution offluoroscein-5-isothiocyanate (23.4 mg, 60 μmol) anddiisopropylethylamine (11.6 mg, 15.7 μL, 90 μmol) in DMF (1.5 mL) for 12hours. The resin was washed thoroughly (NMP/ether/NMP), and treated witha solution of Tl(TFA)₃ (18.7 mg, 34.5 μmol) in DMF (1.5 mL) at 4° C. forthree hours. The resin was washed after this treatment, and treated witha cocktail of TFA/TIS/water (95/2.5/2.5, 2.0 mL) for two hours. Thecrude peptide was precipitated by adding ether to the cleavage cocktailand purified by preparative HPLC using a Vydac C-18 column.

TMR (tetramethylrhodamine) derivatives were prepared using6-carboxytetramethylrhodamine, succinimidyl ester instead offluoroscein-5-isothiocyanate.

Modification of Fibrin Binding Moiety with Optical Dye. See FIG. 2.

Mass Spectrometry and Kd Data of Structures I-XI: MS data Kd (μM) vs.Compound MS data[(M + 2H)/2]+ (M + H)+ DD(E) @ 24° C. Structure I 972.5N/a .1 Structure II 993 N/a .1 Structure III 1022.3 N/a .1 Structure IV1086.7 N/a .06 Structure V 1050.8 N/a .09 Structure VI 1001.2 N/a .2Structure VII 1029.9 N/a .1 Structure VIII 1094.3 N/a .1 Structure IX1058.6 N/a .1 Structure X N/a 1795 N/a Structure XI N/a 2049 0.09N/a = not available

Example 2 Detection of Fibrin-Optical Agent Complex on a Lesion

Site 2/fibrin: 0.1 mg/mL of fibrinogen was mixed with 0.6 μM of anoptical agent (Structure XI, see FIG. 3) comprising tetramethylrhodamineas the optical dye. The mixture was coated (approximately 4-20 μL) ontoa glass slide and cross-linking of fibrinogen was initiated with 1.3μg/L of thrombin. Clotting occurred in approximately 15 sec. The slidewas imaged using confocal fluorescence imaging (ex 555 nm, em 575 nm).Fibrin was detected based on the signal of the fibrin-optical agentcomplexes formed by the binding of Structure XI to fibrin on a lesionformed by cross-linking fibrinogen with thrombin.

Site 2/plasma clot: human plasma (platelet rich human plasma) was mixedwith 0.6 μM of an optical agent (Structure XI, see FIG. 3) comprisingtetramethylrhodamine as the optical dye. The mixture was coated onto aglass slide (approximately 4-20 μL) and clotting of plasma was initiatedwith 1.3 μg/L of thrombin. Clotting occurred within 15 sec. The slidewas imaged using confocal fluorescence imaging (ex 555 nm, em 575 nm).Fibrin was detected based the signal of the fibrin-optical agentcomplexes formed by the binding of Structure XI to fibrin on a lesion(plasma clot) formed by clotting human plasma with thrombin.

Site 1/fibrin: 0.1 mg/mL of fibrinogen was mixed with 0.6 μM of anoptical agent (Structure X) comprising tetramethylrhodamine as theoptical dye. The mixture was coated (approximately 4-20 μL) onto a glassslide and cross-linking of fibrinogen was initiated with 1.3 μg/L ofthrombin. Clotting occurred in about 15-20 seconds. The slide was imagedusing confocal fluorescence imaging (ex 555 nm, em 575 nm). Fibrin isdetected based on the signal of the fibrin-optical agent complexesformed by the binding of Structure X to fibrin on a lesion formed bycross-linking fibrinogen with thrombin.

In other examples, the optical agent was added in approximatelystoichiometric amount to fibrinogen after the clotting of the fibrinogenhad occurred on the surface of a slide. The optical agent was addedafter waiting a period of 10 times the clotting period (e.g., 150 sec.)by layering the solution over the clot on the slide and covering with acover slip.

Example 3 Treatment of a Lesion

A guinea pig (Harley, male) is anaesthetized. An incision is made in theabdomen and the inferior vena cava (IVC) is isolated. The vessel isallowed to recover for 10 mins. A 1 cm portion of the IVC is clamped andhuman thrombin (50 μL, 4 units) is injected into the vessel to promotethrombus formation. The lower clamp is opened and closed to allowpartial blood flow to the segment. After 2-3 mins., the clips areremoved. The thrombus is allowed to age in the animal for 30 mins. Atthis point, the optical agent is administered at a dose of 0.02 μmol/kg,via injection into the jugular vein. After 30 mins., a catheter with anoptical fluorescence detector is inserted into the IVC and the thrombusvisualized by detecting the fluorescence signal emitted by thefibrin-optical agent complexes on the thrombus. Tissue plasminogenactivator (tPA) is delivered through the catheter and the opticalfluorescence signal decreases, indicating clot dissolution and lysis.TNKASE™ (Tenecteplase) is a commercially approved tissue plasminogenactivator (tPA) produced by recombinant DNA technology and sold byGenetech. The drug is administered intravenously at a dose of 30-50 mg,depending on patient weight.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method for treating an intravascular lesion in a patientcomprising: a) administering an optical agent, wherein the optical agentcomprises a fibrin binding moiety and an optical dye, wherein theoptical agent forms a fibrin-optical agent complex at the site of thelesion; b) detecting a signal from the fibrin-optical agent complexusing a device inserted near the lesion; c) obtaining data about thelesion based on the signal from the fibrin-optical agent complex; and d)delivering a therapy to at least a portion of the lesion based on theobtained data.
 2. The method of claim 1, wherein the fibrin bindingmoiety comprises a peptide.
 3. The method of claim 2, wherein theoptical dye is covalently bound to the N-terminal amino acid of thepeptide.
 4. The method of claim 2, wherein the optical dye is covalentlybound to the N-terminal amino acid of the peptide via a linker.
 5. Themethod of claim 2, wherein the fibrin binding moiety comprises the aminoacid sequence Cys-Asp-Tyr-Tyr-Gly-Thr-Cys (SEQ ID NO:1).
 6. The methodof claim 2, wherein the fibrin binding moiety comprises the amino acidsequence Cys-Pro-Tyr-Xaa-Leu-Cys (SEQ ID NO:2), wherein Xaa is Gly orAsp.
 7. The method of claim 2, wherein the fibrin binding moietycomprises the amino acid sequence Cys-Hyp-Tyr(3×)-Xaa-Leu-Cys (SEQ IDNO:3), wherein 3× is selected from the group consisting of halogen,nitro-, and a trifluoromethyl group at the 3 position of the benzyl ringof the tyrosine, and wherein Xaa is Gly or Asp.
 8. The method of claim2, wherein the fibrin binding moiety comprises the amino acid sequencePhe-His-Cys-Hyp-Tyr(3-I)-Asp-Leu-Cys-His-Ile-Leu (SEQ ID NO:4).
 9. Themethod of claim 3, wherein the N-terminal amino acid is selected fromthe group consisting of β-alanine, 6-aminohexanoic acid, and lysine. 10.The method of claim 2, wherein the optical dye is covalently bound tothe C-terminal amino acid of the peptide.
 11. The method of claim 2,wherein the optical dye is covalently bound to the C-terminal amino acidof the peptide via a linker.
 12. The method of claim 3, wherein theC-terminus of the peptide is capped as a C-terminal amide.
 13. Themethod of claim 3, wherein the C-terminus of the peptide is capped witha non-optical moiety.
 14. The method of claim 3, wherein the C-terminalamino acid is in the D-configuration.
 15. The method of claim 1, whereinthe optical dye is selected from the group consisting of fluorescein,rhodamine, hematoporphyrin, fluoresdamine, indocyanine,tetramethylrhodamine, Cosin, erythrosine, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue, TexasRed, and derivatives thereof.
 16. The method of claim 1, wherein theoptical agent is selected from the group consisting of:


17. The method of claim 1, wherein the lesion is selected from the groupconsisting of a thrombus, a clot, an atherosclerotic plaque, and anembolus.
 18. The method of claim 1, wherein the lesion comprises fibrinthat is exposed to blood flowing in the blood vessel.
 19. The method ofclaim 1, wherein the fibrin-optical agent complex has a dissociationconstant value of less than about 10 μM.
 20. The method of claim 1,wherein the fibrin-optical agent complex has a dissociation constantvalue of less than about 5 μM.
 21. The method of claim 1, wherein thefibrin-optical agent complex has a dissociation constant value of lessthan about 1 μM.
 22. The method of claim 1, wherein the fibrin-opticalagent complex has a dissociation constant value of less than about 0.3μM.
 23. The method of claim 1, wherein the optical agent is administeredorally or parenterally.
 24. The method of claim 23, wherein theparenteral administration is intravenous, intraarterial, interstitial,intrathecal, subcutaneous, or intracavity administration.
 25. The methodof claim 1, wherein the device comprises a catheter and an opticaldetector.
 26. The method of claim 25, wherein the optical detector is afluorescence emission detector.
 27. The method of claim 25, wherein thedevice further comprises an excitation source.
 28. The method of claim25, wherein the device is inserted near the lesion in a cavity, atissue, an interstitial space, or a blood vessel.
 29. The method ofclaim 25, wherein the device is inserted in the same blood vessel as thelesion.
 30. The method of claim 1, wherein the device is capable ofdelivering the therapy to at least a portion of the lesion.
 31. Themethod of claim 1, wherein the therapy comprises a thrombolytic agent.32. The method of claim 31, wherein the thrombolytic agent is selectedfrom the group consisting of tissue plasminogen activator,streptokinase, antistreplase, and urokinase.
 33. The method of claim 31,wherein the thrombolytic agent is administered intravenously at a siteremote from the lesion.
 34. The method of claim 31, wherein thethrombolytic agent is delivered to at least about 90% of the surface ofthe lesion.
 35. The method of claim 31, wherein the thrombolytic agentis delivered to at least about 50% of the surface of the lesion.
 36. Themethod of claim 31, wherein the thrombolytic agent is delivered to about10% of the surface of the lesion.
 37. The method of claim 1, wherein thetherapy comprises mechanical manipulation of the lesion.
 38. The methodof claim 37, wherein the mechanical manipulation is selected from thegroup consisting of balloon angioplasty and laser ablation of thelesion.
 39. The method of claim 1, further comprising e) detecting thesignal from the fibrin-optical agent complex during the delivery of thetherapy.
 40. The method of claim 39, further comprising f) stopping thedelivery of the therapy when the signal of the fibrin-optical agentcomplex decreases to a predetermined value.
 41. The method of claim 40,wherein the therapy is stopped when the signal of the fibrin-opticalagent complex is less than about 90% of the signal before delivery ofthe therapy.
 42. The method of claim 40, wherein the therapy is stoppedwhen the signal of the fibrin-optical agent complex is less than about50% of the signal before delivery of the therapy.
 43. The method ofclaim 40, wherein the therapy is stopped when the signal of thefibrin-optical agent complex is less than about 10% of the signal beforedelivery of the therapy.
 44. The method of claim 10, wherein theN-terminus of the peptide is alkylated.
 45. The method of claim 10,wherein the N-terminal amino acid is in the D-configuration.
 46. Acomposition comprising an optical agent, wherein the optical agentcomprises an optical dye covalently linked to the N-terminus of apeptide fibrin binding moiety (FBM) via a linker, said optical agenthaving the general formula:


47. The composition of claim 46, wherein the optical agent is selectedfrom the group consisting of:

and a pharmaceutically acceptable salt thereof.
 48. A formulationcomprising the composition of claim 47, wherein the formulationcomprises at least one ingredient selected from the group consisting ofsolubilizing agents, excipients, carriers, adjuvants, vehicles,preservatives, a local anesthetic, flavorings, and colorings
 49. A kitcomprising the composition of claim
 47. 50. A method to treat a thrombusin a blood vessel in a patient, said method comprising: a) administeringan optical agent, said agent having the structure:

to form a fibrin-optical agent complex; b) inserting a catheter in theblood vessel having said thrombus to obtain information about saidthrombus, said information based on detecting a fluorescence emissionsignal of said fibrin-optical agent complex; and c) delivering with saidcatheter a thrombolytic therapy comprising tissue plasminogen activator(tPA) based on said information to about 90% of said thrombus so thatthe size of said thrombus is reduced.